Ibuprofen Rescues Mutant Cystic Fibrosis Transmembrane
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Journal of Cystic Fibrosis 14 (2015) 16–25 www.elsevier.com/locate/jcf Original Article Ibuprofen rescues mutant cystic fibrosis transmembrane conductance regulator trafficking ⁎ Graeme W. Carlile a, ,1, Renaud Robert b,1, Julie Goepp b, Elizabeth Matthes b, Jie Liao b, Bart Kus c, Sean D. Macknight a, Daniela Rotin c, John W. Hanrahan b, David Y. Thomas a a Cystic Fibrosis Translational Research Center, Dept. of Biochemistry, McGill University, Montreal, Quebec H3G1Y6, Canada b Cystic Fibrosis Translational Research Center, Dept. of Physiology, McGill University, Montreal, Quebec H3G1Y6, Canada c Hospital for Sick Children, Dept. of Biochemistry, University of Toronto, Ontario M5G 1X8, Canada Received 18 December 2013; recieved in revised form 27 May 2014; accepted 1 June 2014 Available online 25 June 2014 Abstract Background: Small molecules as shown by VX809 can rescue the mislocalization of F508del-CFTR. The aim of this study was to identify correctors with a clinical history and their targets of action. Methods: CFTR correctors were screened using two F508del-CFTR expressing cell based HTS assays. Electrophysiological studies using CFBE41o− and HBE cells and in-vivo mouse assays confirmed CFTR rescue. The target of action was attained using pharmacological inhibitors and siRNA to specific genes. Results: Ibuprofen was identified as a CFTR corrector. Ibuprofen treatment of polarized CFBE41o− monolayers increased the short-circuit current (Isc) response to stimulation. In vivo CF mice treatment with ibuprofen restored the CFTR trafficking. SiRNA knock down of cyclooxygenase expression caused partial F508del-CFTR correction. Conclusion: These studies show that ibuprofen is a CFTR corrector and that it causes correction by COX-1 inhibition. Hence ibuprofen may be suitable to be part of a future CF combination therapy. © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; NSAID; Protein folding; Protein trafficking 1. Introduction is retained in the ER prior to degradation by the ubiquitin proteasome system [2,3]. However, if cells are incubated at Cystic fibrosis (CF) is a lethal autosomal recessive disease reduced temperature a small portion of newly synthesized triggered by mutations in the gene encoding the CF transmem- F508del-CFTR does traffick to the cell surface where it is a brane conductance regulator protein [1]. The most common partially functional anion channel [3,4]. disease-associated mutation is a deletion of the phenylalanine The ability to rectify the location of F508del-CFTR has residue at position 508 (F508del-CFTR) with approximately heightened the interest in drug development for this purpose. 70% of all CF patients being homozygous for this mutation. Several groups undertook high throughput-screening (HTS) The F508del-CFTR mutation results in a misfolded protein that projects to identify small molecules that correct F508del-CFTR trafficking [5–7] with Vx-809 (ivacaftor) the only drug that has completed monotherapy testing in the clinic [8]. ⁎ Corresponding author at: Biochemistry Department, McIntyre Medical Sciences Our goal here is to test known drugs that were developed Building, McGill University, 3655 Promenade Sir William Osler, Montreal, QC H3G 1Y6, Canada. Tel.: +1 514 398 1341; fax: +1 514 398 7384. for other indications for their ability to correct F508del-CFTR E-mail address: [email protected] (G.W. Carlile). trafficking. Such compounds have known safety and bioavail- 1 G.W.C and R.R. contributed equally to this work. ability hence potentially reducing the time needed for pre-clinical http://dx.doi.org/10.1016/j.jcf.2014.06.001 1569-1993/© 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. G.W. Carlile et al. / Journal of Cystic Fibrosis 14 (2015) 16–25 17 development and accelerating their approval for clinical use. One 2.4. YFP fluorescence assay compound we identified was ibuprofen. Ibuprofen was first investigated as a CF treatment in 1990 and Compounds were counter screened as previously described is currently used in CF patients to reduce extreme inflammation (and supplementary methods) using Human Embryonic Kidney [9]. Ibuprofen was found to significantly slow the decline in cells (HEK293 GripTite™, Invitrogen) stably expressing F508del- FEV1 over a four-year period and this has been confirmed in CFTR that was transfected with a halide sensitive variant of subsequent studies [10–12]. The effect of ibuprofen has been eYFP [6]. attributed to its anti-inflammatory effect based, in part, on early rat model studies of Pseudomonas infection [9]. 2.5. Transfection for non-CF protein diseases We identified ibuprofen in a cell-based HTS assay specifically designed to detect F508del-CFTR trafficking correctors. Here HeLa cells (5.0 × 106 cells/flask) were transfected with 16 μg we evaluated ibuprofen corrector potency for F508del-CFTR of plasmid and 60 μl of fugene HD overnight. The next day cells processing in several in vitro model systems including polarized were transferred to 6 well dishes (1.0–1.2 × 106 cells/well) and epithelial cells, primary human airway epithelial cell monolayers, 24 h later treated with the compound of interest for 24 h [18]. and freshly isolated intestines from CF mice. We found partial CFTR correction in all these systems. Also, in an in-vivo 2.6. Immunoblots F508del-CFTR mouse assay ibuprofen gave correction. Here we further show that the target of action for ibuprofen induced CFTR Immunoblots were used to measure CFTR maturation using correction maybe via inhibition of the COX protein family a mouse monoclonal anti-CFTR primary antibody (24-1; R&D particularly COX-1. Hence the CF patient benefit of ibuprofen Systems USA. Cat. MAB25031) and a secondary antibody, HRP- treatment is not only due to reducing inflammation but also due to conjugated anti-mouse antibody (Amersham). CFTR correction. 2. Methods 2.7. Iodide efflux assay 2.1. Materials used Iodide efflux was used to measure CFTR functionality in BHK cells as described in supplementary methods and previously Four specific cyclooxygenase (COX) inhibitors were [5]. used; the COX1 inhibitors Sc560 (5-(4-chlorophenyl)-1- (4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole) [13] 2.8. Voltage-clamp of CFBE41o− cell monolayers and TFAP (N-(5-amino-2-pyridinyl)-4-trifluoromethylbenzamide) − [14] the COX2 inhibitors DuP 697 (5-bromo-2-(4-fluorophenyl)- Short-circuit current (Isc) was measured across CFBE41o cell 3-(4-(methylsulfonyl) phenyl)-thiophene) [15] and NPIMA monolayers in Ussing chambers as stated previously [6] and in (N-(3-pyridyl)-indomethacin amide) [16] (all from Cayman the supplementary materials. Chem.). 2.9. CF mice 2.2. Cell culture Homozygous F508del-CFTR mutant mice (Cftrtm1Eur; [6]) The CFBE cell line used is the CFBE41o− derived from and non-CF littermate controls on a FVB background were used. CF patients bronchial epithelial cells and stably infected with Canadian Institutes of Health Research (CIHR) guidelines were TranzVector lentivectors containing either wt or F508del-CFTR. followed and approved by the McGill University Animal Care They were kindly provided by J.P. Clancy (University of Alabama, Committee. Mice were genotyped by PCR [6]. Compounds were Birmingham, USA) [17]. Wild-type V2R and V2R-V206D were tested in-vivo and ex-vivo: as described previously [6] (and in provided by Dr. Peter Deen (NCMLS, The Netherlands). HEK supplementary methods). cells stably expressing HA-tagged hERG G601S or wild-type hERG were given by Eckhard Ficker (Case Western Reserve University U.S.A.) Flag-tagged hamster SUR1 both the wild-type 2.10. High-throughput FACS assay for siRNA treatment and A116P mutant form and rat Kir6.2 plasmids were given by Show-Ling Shyng (Oregon Health and Science University) and HEK293 Flp-In T-Rex cells were utilized as discussed in were reported previously [18]. the supplementary materials and previously [5] to measure the increase in surface CFTR upon siRNA treatment. 2.3. HTS protocol 2.11. RNA extraction and quantitative real-time RT-PCR Screening was performed as described previously (and supplementary methods) using BHK cells that express F508del- Total RNA was extracted and real-time PCR assays were CFTR with 3 tandem hemagglutinin-epitope tags (3HA) in the performed as described previously and in supplementary materials fourth extra-cellular loop [19]. [6]. 18 G.W. Carlile et al. / Journal of Cystic Fibrosis 14 (2015) 16–25 2.12. Statistics (at 10 μM and 320 μM) to 14 ± 1.2% of the amount of CFTR in the lane. In comparison VRT-325 (10μM) had 26% of its CFTR Statistical analysis was performed as described previously expressed in the band C form. and in supplementary materials [6]. To determine if the corrected F508del-CFTR was functional it was measured using an automated iodide efflux assay. Ibuprofen 3. Results treatment (10 μM) for 24 h caused recovery of halide efflux responses to 10 μMforskolin+50μMgenistein(Fig. 3A). 3.1. Ibuprofen partially corrects the F508del-CFTR trafficking Correction was less robust than VRT-325 treatment or in cells defect expressing wild-type CFTR, consistent with the HTS assays and immunoblots. Ibuprofen correction potency and dynamics were Ibuprofen was identified as a CFTR corrector from a screen tested (Fig. 3B, C). Doses between 10 nM and 500 μMof of 3200 known drugs from commercial compound libraries ibuprofen caused a significant F508del-CFTR iodide efflux (Supplemental Table 1 and supplementary information) (Fig. 1A). restoration peaking at 10 μM, consistent with the HTS assay for Ibuprofen gave a 25.7 ± 1.6% increase in cell surface F508del- protein trafficking. Ibuprofen gave a significant response after CFTR signal as compared to wild type signal (Fig. 1B). By treatment with a single dose of 10 μM for 18 to 48 h. comparison, the known corrector VRT-325 gave a 39.5 ± 1.3% increase.